The invention relates to methods of identifying and using a compound that is an agonist to a peptide hormone receptor.
Peptide hormone receptors are important targets for drug research because a considerable number of diseases and other adverse effects result from abnormal receptor activity. One peptide hormone of interest, cholecystokinin (CCK), is a neuropeptide with two distinct receptors: CCK-A and CCK-B/gastrin (Vanderhaeghen et al., Nature, 257:604-605, 1975; Dockray, Nature, 264:568-570, 1976; Rehfeld, J. Biol. Chem., 253:4022-4030, 1978; Hill et al., Brain Res., 526:276-283, 1990; Hill et al., J. Neurosci., 10:1070-1081, 1990; Woodruff et al., Neuropeptides, (Suppl.) 19:57-64, 1991). The peripheral type receptor, the CCK-A receptor, is located in discrete brain nuclei and, in certain species, the spinal cord, and is also involved in gallbladder contraction and pancreatic enzyme secretion. The CCK-B/gastrin receptor is most abundant in the cerebral cortex, cerebellum, basal ganglia, and amygdala of the brain, in parietal cells of the gastrointestinal tract, in ECL cells, as well as in kidney cells. CCK-B receptor antagonists have been postulated to modulate anxiety, panic attacks, analgesia, and satiety (Ravard et al., Trends Pharmacol. Sci., 11:271-273, 1990; Singh et al., Proc. Natl. Acad. Sci. U.S.A., 88:1130-1133, 1991; Faris et al., Science, 219:310-312, 1983; Dourish et al., Eur. J. Pharmacol., 176:35-44, 1990; Wiertelak et al., Science, 256:830-833, 1992; Dourish et al., Science, 245:1509-1511, 1989).
Applicants have developed a systematic screening assay for identifying an agonist specific for a peptide hormone receptor, e.g., a peptide, peptoid, or non-peptide agonist. The assay is based on applicants"" recognition that a peptide hormone receptor able to amplify the intrinsic activity of a ligand, e.g., a constitutively active peptide hormone receptor, is useful as a screening vehicle for identifying a receptor-specific agonist. A receptor with a signaling activity higher than the corresponding human wild-type basal level of signaling activity is further useful for detecting the reduction in signaling activity induced by an inverse agonist. In both cases, the receptor amplifies the signal generated when the ligand interacts with its receptor, relative to the signal generated when the ligand interacts with a human wild-type receptor. Thus, forms of a receptor with the ability to amplify receptor signaling are useful for efficiently screening positive and inverse agonists to the corresponding human wild-type form of the receptor.
Accordingly, the invention features a method for determining whether a candidate compound is an agonist of a peptide hormone receptor. In this method, a candidate compound is exposed to a form of the peptide hormone receptor which has a greater, or an enhanced, ability to amplify the intrinsic activity of an agonist (hereafter an xe2x80x98enhanced receptorxe2x80x99). The second messenger signaling activity of the enhanced receptor is measured in the presence of the candidate compound, and compared to the second messenger signaling activity of the enhanced receptor measured in the absence of the candidate compound. A change in second messenger signaling activity indicates that the candidate compound is an agonist. For example, an increase in second messenger signaling activity indicates that the compound is either a full or partial positive agonist; a decrease in second messenger signaling activity indicates that the compound is an inverse (also termed a xe2x80x98negativexe2x80x99) agonist. The method can further comprise using the agonist to treat or to prevent a physiological disorder involving a peptide hormone receptor by administering to a mammal the identified agonist in an agonist-effective amount.
By xe2x80x9cintrinsic activityxe2x80x9d is meant the ability of a ligand to activate a receptor, i.e., to act as an agonist. By xe2x80x98amplifyxe2x80x99 is meant that the signal generated when the ligand interacts with the enhanced receptor is either higher for a positive agonist, or lower for an inverse agonist, than the signal produced when the same ligand interacts with a corresponding non-enhanced receptor, e.g., a wild-type human receptor. A xe2x80x98non-enhanced receptor,xe2x80x99 for the purposes of this invention, is a wild-type human receptor for the peptide hormone of interest. By xe2x80x9ccorrespondingxe2x80x9d is meant the same type of peptide hormone receptor albeit in another form, e.g., a constitutively active mutant receptor. By way of example, the corresponding wild-type form of a constitutively active mutant CCK-B/gastrin receptor would be a wild-type CCK-B/gastrin receptor; the human CCK-B/gastrin receptor is the corresponding human form of the rat CCK-B/gastrin receptor.
An xe2x80x9cagonist,xe2x80x9d as used herein, includes a positive agonist, e.g., a full or a partial positive agonist, or a negative agonist, i.e., an inverse agonist. An agonist is a chemical substance that combines with a receptor so as to initiate an activity of the receptor; for a peptide hormone receptor, the agonist preferably alters a second messenger signaling activity. A positive agonist is a compound that enhances or increases an activity, e.g., a second messenger signaling activity, of a receptor. A xe2x80x9cfull agonistxe2x80x9d refers to an agonist capable of activating the receptor to the maximum level of activity, e.g., a level of activity which is induced by a natural, i.e., an endogenous, peptide hormone. A xe2x80x9cpartial agonistxe2x80x9d refers to a positive agonist with reduced intrinsic activity relative to a full agonist. As used herein, a xe2x80x9cpeptoidxe2x80x9d is a peptide-derived partial or full agonist (Horwell et al., Eur. J. Med. Chem., 30 Suppl.:537S-550S, 1995; Horwell et al., J. Med. Chem., 34:404-14, 1991).
An xe2x80x9cinverse agonist,xe2x80x9d as used herein, has a negative intrinsic activity, and reduces the receptor""s signaling activity relative to the signaling activity of the wild-type receptor measured in the absence of the inverse agonist. In contrast, an xe2x80x9cantagonist,xe2x80x9d as used herein, refers to a chemical substance that inhibits the ability of an agonist to increase or decrease receptor activity. A xe2x80x98full,xe2x80x99 or xe2x80x98perfectxe2x80x99 antagonist has no intrinsic activity, and no effect on the receptor""s basal activity (FIG. 1). Peptide-derived antagonists are, for the purposes herein, considered to be non-peptide ligands.
A diagram explaining the difference between full and partial agonists, inverse agonists, and antagonists is shown in FIG. 1 (see also Milligan et al., TIPS, 16:10-13, 1995). In FIG. 1, the position of the equilibrium between an inactive state R and an active state R* varies with individual receptors and is altered by the presence of receptor ligands. Agonists function by stabilizing R* while inverse agonists preferentially stabilize R. A continuum of ligands between full agonists (at the extreme right-hand side of the see-saw as these move the equilibrium furthest to the right) and full inverse agonists (at the extreme left-hand side of the see-saw) is expected to exist. Antagonists, which do not alter the position of the equilibrium, define the position of the fulcrum. An antagonist is, e.g., a competitive or a non-competitive inhibitor.
Examples of peptide hormone receptor specific peptide and non-peptide agonists useful in the screening assay of the invention are described below. Non-peptide ligands include, but are not limited to, benzodiazepines and derivatives thereof, e.g., azabicyclo[3.2.2]nonane benzodiazepine (xe2x80x9cL-740,093xe2x80x9d; see Castro Pineiro et al., WO 94/03437; Castro Pineiro et al., U.S. Pat. No. 5,521,175). L-740,093 S and L-740,093 R refer to the S-enantiomer and the R-enantiomer of L-740,093, respectively. Where the peptide hormone receptor is a CCK-A receptor or a CCK-B/gastrin receptor, useful peptide agonists include, but are not limited to, gastrin (e.g., sulphated (xe2x80x9cgastrin IIxe2x80x9d) or unsulphated (xe2x80x9cgastrin Ixe2x80x9d) forms of gastrin-17, or sulphated or unsulphated forms of gastrin-34), or cholecystokinin (CCK) (e.g., sulfated CCK-8 (CCK-8s), unsulphated CCK-8 (CCK-8d), CCK-4, or pentagastrin). Full agonists of the CCK-B/gastrin receptor include, but are not limited to, CCK-8s, and more preferably gastrin (gastrin I).
An enhanced receptor may, but does not always, have a higher basal activity than the basal activity of a corresponding human wild-type receptor. Methods for measuring the activity of an enhanced receptor relative to the activity of a corresponding wild-type receptor are described and demonstrated below. Examples of enhanced receptors include synthetic mutant receptors, e.g., constitutively active mutant receptors; other mutant receptors with normal basal activity which amplify the intrinsic activity of a compound; naturally-occurring mutant receptors, e.g., those which cause a disease phenotype by virtue of their enhanced receptor activity, e.g., a naturally-occurring constitutively active receptor; and either constitutively active or wild-type non-human receptors, e.g., rat, mouse, mastomys, Xenopus, or canine receptors or hybrid variants thereof, which amplify an agonist signal to a greater extent than does the corresponding wild-type human receptor.
Further examples of peptide hormone receptors useful in the screening assay of the invention include, but are not limited to, receptors specific for the following peptide hormones: amylin, angiotensin, bombesin, bradykinin, C5a anaphylatoxin, calcitonin, calcitonin-gene related peptide (CGRP), corticotropin releasing hormone (CRH), chemokines, cholecystokinin (CCK), endothelin, erythropoietin (EPO), follicle stimulating hormone (FSH), formyl-methionyl peptides, galanin, gastrin, gastrin releasing peptide, glucagon, glucagon-like peptide 1, glycoprotein hormones, gonadotrophin-releasing hormone, leptin, luteinizing hormone (LH), melanocortins, neuropeptide Y, neurotensin, opioid, oxytocin, parathyroid hormone, secretin, somatostatin, tachykinins, thrombin, thyrotrophin, thyrotrophin releasing hormone, vasoactive intestinal polypeptide (VIP), and vasopressin. An enhanced receptor can further embrace a single transmembrane domain peptide hormone receptor, e.g., an insulin receptor.
The invention also features a method of isolating a form, e.g., a mutant form, of a peptide hormone receptor that is suitable for detecting agonist activity in a ligand, e.g., a peptide, peptoid, or non-peptide ligand. The method involves (a) exchanging a region of a functional domain of a first peptide hormone receptor with a corresponding region of a functional domain of a second peptide hormone receptor; and (b) measuring the ability of the first peptide hormone receptor to amplify an agonist signal relative to a corresponding wild-type human receptor. The functional domain can be an intracellular loop, parts of a transmembrane domain adjacent to an intracellular loop, a transmembrane domain, a region of a transmembrane domain distal to an intracellular loop, or an extracellular loop. A level of amplification by the first peptide hormone receptor that is greater than the level of amplification by the wild-type human receptor indicates that the first peptide hormone receptor is suitable for detecting agonist activity in a non-peptide ligand. The corresponding region can be between one and ten amino acids, e.g., a block of five to ten amino acids, or up to thirty or a hundred amino acids in length. The first and second peptide hormone receptors are preferably linked to different second messenger pathways. Those skilled in the art know which particular amino acids of the peptide hormone receptors are considered to be within extracellular, intracellular (cytoplasmic), or transmembrane regions of the receptor. For example, extracellular, intracellular, and transmembrane regions of the CCK-B/gastrin receptor are determined by sequence alignment with other receptors (FIGS. 2A and 2B), or by hydropathy analysis (Baldwin, EMBO J., 12:1693-1703, 1993). Conformation receptor modelling is described further below.
Another method of isolating a form of a peptide hormone receptor suitable for detecting agonist activity in a non-peptide ligand involves (a) constructing a series of mutant forms of the receptor by replacing an original amino acid with another amino acid, i.e., a replacement amino acid; and (b) measuring the ability of the resulting peptide hormone receptor mutant form to amplify an agonist signal relative to the level of amplification by a corresponding wild-type human receptor. An amplification in the peptide hormone receptor mutant form that is greater than the level of amplification of the corresponding wild-type human receptor indicates that the mutant form is suitable for detecting agonist activity in a non-peptide ligand. The replaced amino acid can lie in an intracellular domain of the receptor or in a region of a transmembrane domain flanking an intracellular portion of the receptor, e.g., the intracellular domain-proximal half of the transmembrane domain, or within, e.g., 8 or 10 amino acids of the intracellular domain. The replacement amino acid can be of the same type in each of the mutant constructs; alternatively, various types of amino acids can be substituted at random. The replacement amino acid can be of the same charge, or of a different charge, than the original amino acid, e.g., a negative amino acid can be exchanged for a positive amino acid, a positive amino acid can be exchanged for a negative amino acid, or a positive or negative amino acid can be exchanged for a neutral amino acid. Preferably, the replacement amino acid is glutamine, glutamic acid, aspartic acid, or serine.
Also embraced are the various mutant peptide hormone receptors disclosed herein, and their respective nucleic acid coding sequences. Mutant peptide hormone receptors of the invention include, but are not limited to, the CCK-A receptor MHA21/35, and the mutant CCK-B/gastrin receptors MH40, MH128, MH156, MH162, MH31, MH131, MH13, MH130, MH129, and MH72. Plasmid manipulation, storage, and cell transformation can be performed by methods known to those of ordinary skill in the art. See, e.g., Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1988, 1995.
With an efficient and rapid assay for identifying an agonist specific for a given peptide hormone receptor, those skilled in the art can identify agonists to serve as lead compounds for further pharmaceutical research. In particular, systematic chemical modifications can be made, and their effects can be further assessed using an enhanced receptors according to the method of the invention. By following such a development strategy the intrinsic activity of new agonists can be optimized so as to be useful therapeutically against a disease involving a peptide hormone receptor.
Knowing that a particular ligand functions as a positive or inverse agonist, as opposed to an antagonist, facilitates identifying which ligand species is most likely to achieve a given physiological effect, or to achieve a physiological effect absent an unwanted side effect. Thus, the invention further features a method for the treatment or prevention of a physiological disorder involving a peptide hormone receptor that includes administering to a mammal, e.g., a human, a ligand which acts as an agonist on a peptide hormone receptor. The ligand is administered in an agonist-effective amount, i.e., in an amount that has a full or a partial inverse, or a full or a partial positive, agonist effect on a peptide hormone receptor. The peptide hormone receptor can be, but need not be limited to, a receptor specific for one of the following peptide hormones: amylin, angiotensin, bombesin, bradykinin, c5a anaphylatoxin, calcitonin, calcitonin-gene related peptide (CGRP), corticotropin releasing hormone (CRH), chemokines, cholecystokinin (CCK) (e.g., the CCK-A or the CCK-B/gastrin receptor), endothelin, erythropoietin (EPO), follicle stimulating hormone (FSH), formyl-methionyl peptides, galanin, gastrin, gastrin releasing peptide, glucagon, glucagon-like peptide 1, glycoprotein hormones, gonadotrophin-releasing hormone, insulin, leptin, luteinizing hormone (LH), melanocortins, neuropeptide Y, neurotensin, opioid, oxytocin, parathyroid hormone, secretin, somatostatin, tachykinins, thrombin, thyrotrophin, thyrotrophin releasing hormone, vasoactive intestinal polypeptide (VIP), and vasopressin.
An inverse agonist is particularly useful for treating or preventing a physiological disorder that results from enhanced basal activity of a peptide hormone receptor, e.g., a constitutively active receptor. For example, an inverse agonist is useful for treating a neoplasm that results from, or is sustained or aggravated by, enhanced activity of a peptide hormone receptor, e.g., a naturally-occurring peptide hormone receptor. Examples include, but are not limited to, a CCK-B/gastrin related tumor, e.g., a neuroendocrine tumor, Zollinger Ellinger Syndrome, or a gastric carcinoid tumor; a TSH-related tumor; multiple endocrine neoplasia type I; a lung tumor, e.g., a small-cell carcinoma; a brain tumor, e.g., a brain tumor involving CCK; a kidney tumor, e.g., hypernephroma or renal cell carcinoma. Agonists are particularly useful for treating a primary tumor, e.g., a tumor in a tissue that expresses a peptide hormone receptor, e.g., a tumor in the pancreas, the pituitary, or the adrenal gland.
In other embodiments of the invention, an inverse agonist of the LH receptor can be a useful compound for treating or preventing precocious puberty; an inverse agonist of the FSH receptor can be a useful compound for treating or preventing infertility; an inverse agonist of the TSH receptor can be a useful compound for treating or preventing thyroid adenomas. An inverse or positive agonist of the G-LP1 receptor can be a useful compound for treating or preventing obesity or diabetes, e.g., type-I or type-II diabetes.
Non-peptide agonists are further useful for treating or preventing a disorder involving the gastrointestinal tract, or a disorder involving, e.g., sleep, anxiety, panic, appetite regulation, stress, or pain, or a disorder discussed below.
A candidate compound useful in a method of treatment or prevention of a physiological disorder of the invention is a ligand that binds with specificity to a peptide hormone receptor, e.g., a peptide, peptoid, or non-peptide ligand, preferably a non-peptide ligand. A candidate compound is shown to be a positive or inverse agonist by a screening assay disclosed herein. Exemplified candidate compounds include the peptoid compounds ((see, e.g., Horwell et al., Eur. J. Med. Chem., 30 Suppl.:537S-550S, 1995; Horwell et al., J. Med. Chem., 34:404-14, 1991); the dipeptoid analogues of CCK (see, e.g., Horwell et al. J. Med. Chem., 34:404-14, 1991); cyclic nucleotides and modified amino acids (see, e.g., Dethloff et al., Drug Metab., 24:267-93, 1992), the benzodiazepine derivatives, e.g., the compounds described in Bock et al., J. Med. Chem., 33:450-55, 1990, or a derivative thereof. Additional benzodiazepine derivatives having a peptide hormone receptor agonist activity are described in the following patents and patent applications, each of which is hereby incorporated by reference: EPA 167919, EPA 284256, EPA 434360, EPA 434364, EPA 434369, EPA 514125, EPA 51426, EPA 514133, EPA 508796, EPA 508797, EPA 508798, EPA 508799, EPA 523845, EPA 523846, EPA 559170, EPA 549039, EPA 667,334, WO 9211246, WO 93032078, WO 9308175, WO 9307131, WO 9317011, WO 9319053, WO 9308175, WO 9413648, WO 9403437, WO 9611689, and U.S. Pat. No. 5,521,175. Also encompassed are those compounds described in Henke et al., J. Med. Chem., 39:2655-58, 1996; or in Willson et al., J. Med. Chem., 39:3030-34, 1996 (both hereby incorporated by reference) which are shown to be inverse agonists.
As a further example, a benzodiazepine compound useful in a method of treatment or prevention of a physiological disorder resulting from a peptide hormone receptor is a benzodiazepine compound of formula (I): 
wherein:
R1 represents H, C1-6 alkyl optionally substituted by one or more halo, C3-7 cycloalkyl, cyclopropylmethyl, (CH2)rimidazolyl, (CH2)rtriazolyl, CH2))rtetrazolyl (where r is 1, 2 or 3), CH2CO2R11 (where R11 is C1-4alkyl) or CH2CONR6R7 (where R6 and R7 each independently represents H or C1-4alkyl, or R6 and R7 together form a chain CH2p where p is 4 or 5;
R2 represents NHR12 or (CH2)qR13 (where s is 0, 1, 2, or 3);
R3 represents C1-6alkyl, halo or NR6R7, where R6 and R7 are as previously defined;
R4 and R5 each independently represents H, C1-12alkyl optionally substituted by NR9R9xe2x80x2 (R9 and R9xe2x80x2 are as previously defined) or an azacyclic or azabicyclic group, C4-9cycloalkyl optionally substituted by one or more C1-4alkyl groups, C4-9cycloalkylC1-4alkyl optionally substituted in the cycloalkyl ring by one or more C1-4alkyl groups, optionally substituted aryl, optionally substituted arylC1-6alkyl or azacyclic or azabicyclic groups, or R4 and R5 together form the residue of an optionally substituted azacyclic or azabicyclic ring system;
x is 0, 1, 2, or 3;
R12 represent a phenyl or pyridyl group optionally substituted by one or more substituents selected form C1-6alkyl, halo, hydroxy, C1-4alkoxy, (CH2)q-tetrazolyl optionally substituted in the tetrazole ring by C1-4alkyl, (CH2)q-imidazolyl, (CH2)q-triazolyl (qhere q is 0, 1, 2, or 3), 5-hydroxy-4-pyrone, NR6R7, NR9COR11, NR9CONR9xe2x80x2R11 (where R9 and R9xe2x80x2 are each independently H or C1-4alkyl and R11 is as previously defined), SO(C1-6alkyl), SO2(C1-6alkyl), trifluoromethyl, CONHSO2R8, SO2NHCOR8 (where R8 is C1-6alkyl, optionally substituted aryl, 2,2-difluorocyclopropane or trifluoromethyl), SO2NHR10(where R10 is a nitrogen containing heterocycle), B(OH)2, (CH2)qCO2H, where q is as previously defined; or
R12 represents a group: 
xe2x80x83where X1 represents CH or N; W represents CH2 or NR9, where R9 is as previously defined, and W1 represents CH2, or W and W1 each represent O; or
R12 represents phenyl substituted by a group: 
xe2x80x83wherein X2 is O, S or NR9, where R9 is as previously defined; z is a bond, O or S; m is 1, 2 or 3; n is 1, 2, or 3; and y is 0, 1, 2, or 3;
R13 represents a group: 
xe2x80x83where R14 represents H or C1-6alkyl; R15 represents H, C1-6alkyl, halo or NR6R7, where R6 and R7 are as previously defined; and the dotted line represents an optional covalent bond; and pharmaceutically acceptable salts or prodrugs thereof, with the provision that, when NR4R5 represents an unsubstituted azacyclic ring system, R2 does not represent NHR12 where R12 is optionally substituted phenyl or: 
Compounds of formula (I) are intended to embrace all possible racemers and isomers, including optical isomers, and mixtures thereof. Each expression, where occurring more than once in any structure, intended to be independent of its definition elsewhere in the same structure. The present invention includes within its scope prodrugs of the compounds of formula (I) above. In general, such prodrugs will be functional derivatives of the compounds of formula (I) which are readily convertible in vivo into the required compound of formula (I). Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in xe2x80x9cDesign of Prodrugsxe2x80x9d ed. H. Bungaard, Elsevier, 1985.
As used herein, unless otherwise indicated, alkyl means straight or branched chain saturated hydrocarbon; halo includes fluoro, chloro, bromo, and iodo; as used herein, unless otherwise indicated, alkyl means straight or branched chain saturated hydrocarbon; azacyclic means non-aromatic nitrogen-containing monocyclic, and azabicyclic means non-aromatic nitrogen-containing bicyclic; aryl means optionally substituted carbocyclic or heterocyclic aromatic groups, especially phenyl; heteroaryl means aromatic rings preferably having r or 6 ring atoms and containing at least one atom selected from O, S, and N. Compounds of formula (I) are prepared according to the methods of WO 94/03437, hereby incorporated by reference.
By using the screening assay of the invention, those skilled in the art can easily identify which candidate ligand is optimal for therapeutic or preventive use. For example, preferred agonists and their respective derivatives include, but are not limited to, L-740,093 [3(R,S)-Amino-1,3-dihydro-5-((1S,4S)-5-methyl-2,5-diazabicyclo[2,2,1]heptan-2-vl)-2H-1-propyl-1,4-benzodiazepin-2-one]; L-740,093 R [(-)-N-[5-(3-azabicyclo[3.2.2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]-Nxe2x80x2-[3-methylphenyl]urea]; L-740.093 S [(+)-N-(5-(3-azabicyclo[3.2.2]nonan-3-yl)-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]-Nxe2x80x2-[3-methylphenyl]urea]; L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-Nxe2x80x2-(3-methylphenylurea)]; and L-364,718.
Other terms used in the various embodiments of the invention will be understood from the following definitions. For example, by a xe2x80x9cpeptide hormonexe2x80x9d is meant a polypeptide that interacts with a target cell by contacting an extracellular receptor, i.e., a xe2x80x9cpeptide hormone receptor.xe2x80x9d A xe2x80x9cpeptidexe2x80x9d is used loosely herein to refer to a molecule comprised, at least in part, of amino acid residues that are connected to each other by peptide bonds. A xe2x80x9cmutant receptorxe2x80x9d is understood to be a form of the receptor in which one or more amino acid residues in the corresponding receptor which predominates in nature, e.g., in a naturally-occurring wild-type receptor, have been either deleted or replaced with a different type of amino acid residue. By a xe2x80x9cconstitutively active receptorxe2x80x9d is meant a receptor with a higher basal activity level than the corresponding wild-type receptor, where xe2x80x9cactivityxe2x80x9d refers to the spontaneous ability of a receptor to signal in the absence of further activation by a positive agonist. The basal activity of a constitutively active receptor can also be decreased by an inverse agonist. A xe2x80x9cnaturally-occurringxe2x80x9d receptor refers to a form or sequence of the receptor as it exists in an animal, or to a form of the receptor that is synonymous with the sequence known to those skilled in the art as the xe2x80x9cwild-typexe2x80x9d sequence. Those skilled in the art will understand a xe2x80x9cwild-typexe2x80x9d receptor to refer to the conventionally accepted xe2x80x9cwild-typexe2x80x9d amino acid consensus sequence of the receptor, or to a xe2x80x9cnaturally-occurringxe2x80x9d receptor with normal physiological patterns of ligand binding and signaling. A xe2x80x9csecond messenger signaling activityxe2x80x9d refers to production of an intracellular stimulus (including, but not limited to, cAMP, cGMP, ppGpp, inositol phosphate, or calcium ion) in response to activation of the receptor, or to activation of a protein in response to receptor activation, including but not limited to a kinase, a phosphatase, or to activation or inhibition of a membrane channel.
xe2x80x9cSequence identity,xe2x80x9d as used herein, refers to the subunit sequence similarity between two nucleic acid or polypeptide molecules. When a given position in both of the two molecules is occupied by the same nucleotide or amino acid residue, e.g., if a given position (as determined by conventionally known methods of sequence alignment) in each of two polypeptides is occupied by serine, then they are identical at that position. The identity between two sequences is a direct function of the number of matching or identical positions, e.g., if 90% of the positions in two polypeptide sequences are identical, e.g., 9 of 10, are matched, the two sequences share 90% sequence identity. Methods of sequence analysis and alignment for the purpose of comparing the sequence identity of two comparison sequences are well known by those skilled in the art. xe2x80x9cBiological activity,xe2x80x9d as used herein, refers to the ability of a peptide hormone receptor to bind to a ligand, e.g., an agonist or an antagonist, and to induce signaling.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.